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Impact of secondary TCR engagement on the heterogeneity of pathogen-specific CD8+ T cell response during acute and chronic toxoplasmosis

ABSTRACT: Initial TCR-dependent engagement of CD8+ T cells results in T cell expansion, and the early events involved in priming influence the generation of diverse effector and memory populations. During infection, some of these activated T cells re-encounter cognate antigen, but whether this influences phenotypic heterogeneity is unclear. To address this issue, OT-I T cells, which are specific for the SIINFEKL peptide from OVA and express the Nur77-GFP reporter of recent TCR activation, were paired with transgenic T. gondii that express OVA to assess the impact of TCR activation on parasite-specific CD8+ T cell responses.

ORGANISM(S): Mus musculus

PROVIDER: GSE188495 | GEO | 2021/11/11

REPOSITORIES: GEO

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T cell receptor signal strength and antigen affinity differentially regulate memory CD8+ T cell development

Project description:IL-2 inducible tyrosine kinase (Itk) is a Tec family non-receptor tyrosine kinase that is known to regulate T cell receptor signal strength (TcR) and T cell development and differentiation. TcR signal strength and antigen affinity are parameters known to regulate the development of CD8+ T cell memory. However, the intersection between TcR signal strength and antigen affinity on CD8+ memory T cell development remains unclear. Therefore, we compared the transcriptomes of WT and Itk-/- OT-1/Rag-/- CD8+ T cells at day 0 and day 7 following infection with Listeria monocytogenes expressing the SIINFEKL (N4) or SIITFEKL (T4) OVA peptide variant (different affinities for TcR) by RNA sequencing.

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Analysis of the interactomes of ZAP70, CD247 and SLP76 upon stimulation of murine CD8+ T cells with ligands of different affinity to the TCR

Project description:T cells have the remarkable ability to sense and discriminate between a wide range of antigenic peptides bound to major histocompatibility complex molecules (pMHC). Here, we use interactomics to monitor in a time dependent manner the early molecular events taking place upon stimulation of murine CD8+ T cells with ligands of different affinity to the TCR. We used the mouse OT-I model in which T cells specifically recognize the ovalbumin OVA 257-264 peptide (SIINFEKL, also named N4) bound to the MHC-I molecule H-2Kb (pMHC). The OT-I TCR also recognizes altered peptide ligands amongst which SIITFEKL (T4) and SIIGFEKL (G4) with sub-optimal binding capacities (ligand affinity N4>T4>G4). For affinity purification (AP) of signalling complexes, we crossed OT-I TCR transgenic mice onto mice expressing an endogenous tagged (One-Strep-Tag: OST) version of the CD3z (also called CD247), ZAP70 or SLP76 molecules. CD8+ T cells were purified from pooled lymph nodes and spleens, shortly expanded, and were left unstimulated or stimulated with N4, T4 and G4 soluble pMHC tetramers for 30, 120 or 300 s. Complexes formed around either CD247, ZAP70 or SLP76 were then purified and analysed by mass spectrometry. Each AP-MS purification is associated with a corresponding control (purification from OT-I CD8+ T cells expressing only the endogenous version of each bait, cultured and stimulated in the same conditions). The number of replicate biological experiments was n=3 for all time-points, and each sample was analyzed twice by single-run nano LC-MS. In addition, the whole proteome of OT-I cell was analyzed by label-free shotgun proteomics in order to calculate cellular protein abundances for each interactor.

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Gene expression profiles of OT-1 T cells co-cultured with MHC-I Negative Tumors and Antigen Loaded Macrophages

Project description:OT-1 T-cells were co-cultured either alone in T-cell media supplemented with IL-2 (50IU/mL IL-2), with OVA loaded macrophages, or with both OVA loaded macrophages and CT2A-TRP2-β2mKO tumor cells. Cells were cultured at a 5:1 T-cell to tumor ratio, and 2:1 T-cell to macrophage ratio. After 24 h of co-culture, CD8 T-cells were FACS sorted, and RNA extracted (RNeasy Mini Kit, Qiagen). RNA was analyzed on an nCounter MAX Analysis System (Nanostring) with the PanCancer Immune profiling panel (Nanostring) according to manufacturer instructions. Expression dat OT-1 T-cells were isolated from OT-1 mice by culturing OT-1 splenocytes in T-cell media supplemented with 50IU/mL IL-2 and 1 μM OVA SIINFEKL peptide (Anaspec) for 48 h. Cells were purified for CD8 T-cells as described above and subsequently cultured in TCM with 50IU/mL IL-2, splitting every 24h for a total of 4 days.

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Project description:Activation of CD8+ T cells depends exquisitely on the affinity of the T cell receptor (TCR) for a peptide MHC (pMHC) ligand complex. Here, we activated OT-I transgenic CD8+ T cells with pure peptide and examined early activation responses by single-cell RNA-sequencing. T cells were activated with the high affinity OT-I cognate peptide (N4=SIINFEKL) for 1, 3 or 6 hours, or with reduced affinity peptides (T4=SIITFEKL and G4=SIIGFEKL) or the non-binding peptide (NP68=ASNENMDAM) for 6 hours. Cells were then sorted into 96-well plates by FACS and RNA was sequenced following an adapted Smart-Seq2 protocol.

2018-06-25 | E-MTAB-6051 | biostudies-arrayexpress

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Transcription profiling of mouse splenocytes from OT-1 TCR transgenic mice to assess the effect of IL-12 on CTL gene expression

Project description:The goal was to determine how IL-12 affects gene expression by murine CTL. Experiment Overall Design: Following RBC lysis, splenocytes from OT-1 TCR transgenic mice (5 million/ml) were cultured in six well-plates (5-6ml/well) in IMDM supplemented with 10% FCS and 55Î¼M 2-ME with 1Î¼M SIINFEKL. After 4 days, the cells were harvested by purification with Ficoll-Paque PLUS and placed in fresh medium containing 0 or 20ng/ml rmIL-12. Twenty-four hours later the cells were harvested and used in experiments. The live cells harvested were >98% CD8+VÎ±2+ (OT-1).

2009-02-08 | E-GEOD-13173 | biostudies-arrayexpress

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2013-12-10 | E-GEOD-53200 | biostudies-arrayexpress

Total RNA: 24h activated mouse OT-1 T lymphocytes, WT vs Lck-/- (OFF)

Project description:This experiment aims to study the transcriptome of activated T cells at 24h, using the same biological samples as the polysome profiling in GSE84781. A T cell receptor (TCR) transgenic mouse model (OT-1) posessing a homogenous alpha-beta CD8 T lymphocyte population was used, and cells with intact TCR signalling pathways (WT) were compared to those with defective signal transduction (Lck-/-, OFF). Ex vivo OT-1 T cells were isolated from the superficial cervicals, axillary, brachial, mesenteric, and inguinal lymph nodes and then activated for 24h with cognate peptide antigen SIINFEKL. Total RNA was extracted from the cells, labelled with Cy3, and hybridised onto an Agilent-074809 SurePrint G3 Mouse GE v2 8x60K Microarray.

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Whole-genome microarray analysis of primary, secondary, tertiary and quaternary memory CD8 T cells

Project description:The transcriptome of naive OT-I T cells was compared to memory CD8 T cells after 1, 2, 3, or 4 infection with ovalbumin expressing Listeria monocytogenes (LM-OVA). Naive Thy1.1 OT-I T cells were adoptively transferred into Thy1.2 naive hosts prior to infection with LM-OVA. The resulting memory CD8 T cell population was again adoptively transferred into naive hosts and the recipient mice were again infected with LM-OVA. The adoptive transfer was repeated up to four times to generate memory CD8 T cells with up to four consecutive antigen stimulations. Three individual mice were analyzed for each group. For quaternary memory CD8 T cells, spleens from two to three mice were pooled for each sample. Naive OT-I T cells served as control samples. http://dx.doi.org/10.1016/j.immuni.2010.06.014

2010-07-09 | E-GEOD-21360 | biostudies-arrayexpress

Polysome profiling: Mouse OT-1 T cells WT vs Lck-/- (OFF)

Project description:In order to identify gene targets of translational regulation during T cell activation, a polysome analysis was performed in a T cell receptor (TCR) transgenic model (OT-1) possessing a homogenous alpha-beta CD8 T lymphocyte population. Cells with defective TCR signal tranduction (Lck-/-, OFF) were compared to WT cells to visualise targets regulated by TCR signalling. Total cytoplasmic ribonucleoprotein was extracted from ex vivo OT-1 transgenic T lymphocytes stimulated with SIINFEKL peptide antigen for 24h, extracts were fractionated using a sucrose density gradient, and separated into a sub-polysomal and a polysomal fraction. Total RNA from each fraction was extracted, equal volume of RNA from each fraction was individually labelled (Sub-poly: Cy3; Poly: Cy5), and hybridised onto a Agilent SurePrint G3 Mouse GE v2 8x60K Microarray.

2016-11-30 | GSE84781 | GEO

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